PROJECT SUMMARY/ABSTRACT Antiviral vaccines remain the largest segment of the rapidly growing global vaccine market. Despite near-complete eradication of poliovirus through vaccination efforts, continued need for inactivated polio vaccine (IPV) is projected to reach 450 million doses per year worldwide. Among major obstacles to widespread use of the current IPV formulation are its high cost and insufficient potency to induce robust intestinal immunity. Therefore, development of an effective, safe and affordable adjuvant for IPV that would allow reduction of the costly IPV antigen content represents a highly significant unmet need. This Phase I SBIR proposal aims to address this challenge through optimization of a novel adjuvant system combining the immunostimulatory power of the toll-like receptor 5 (TLR5) agonist, entolimod (previously CBLB502), with the well- known adjuvant properties of Alum. Entolimod is being developed for tissue protective (anti- radiation and supportive care in cancer treatment) and anticancer immunotherapeutic applications by Cleveland BioLabs, Inc. (CBLI) and has an established safety profile. Given the demonstrated adjuvant properties of the natural TLR5 agonist (and entolimod parent) flagellin, investigators of this proposal in partnership with CBLI developed an innovative [entolimod+Alum]-based adjuvant system (termed SA702) with advantages in terms of efficacy (combined effect of two adjuvants), versatility (modular platform easily customized for many different antigen types); and decreased futile (adjuvant-directed) immunogenicity. Preliminary studies confirmed that immunization of mice with IPV (10% human dose) co-adsorbed on SA702 led to induction of increased levels of anti-poliovirus neutralizing antibodies compared to immunization with IPV alone or [IPV+Alum]. Here, we propose further optimization of entolimod as a co-adjuvant through targeting of its residual immunogenicity and its ancillary inflammasome-directed signaling activity (Aim 1) followed by characterization and optimization of the stability, efficacy and safety of SA702-IPV formulations containing the identified optimal entolimod variant (Aim 2). In addition, we will determine the impact of entolimod's residual immunogenicity on adjuvant activity of SA702 via mapping and elimination of mouse T cell epitopes. Together, the deliverables of these aims will define and validate an optimal design for the SA702 platform and a new SA702-IPV formulation and set the stage for Phase II SBIR application and commercialization.